Dr. Guillemin and coworkers at the Salk Institute have recently (Science, 218, 585-587 (Nov. 5, 1982), see also New York Times, Oct. 29, 1982 at page 1, column 2) reported the isolation, synthesis, and biological activity of a group of related substances they have called growth hormone releasing factor (GRF). This factor has been sought after for decades by scientists but such search has been, until now, unrewarding due to the minute quantities in which such substance occurs naturally.
The successful isolation of GRF has been due in part to the discovery of the ectopic production of GRF in large amounts by pancreatic tumors, associated with acromeglay. Three forms of GRF derived from the pancreatic tumor have been observed. These forms consisting of three homologous peptides of 44, 40 and 37 amino acids in length are identical at the amino terminal and differ in the termination point of the carboxyl terminal. The 44 amino acid GRF is further distinguished in having an amide group at the carboxy terminus whereas the other two forms have a free carboxy group at that terminus.
The amidated form of GRF-44 is apparently the parent molecule and has been indicated to process the highest biological activity in vitro. However, all three peptides have been found to be virtually equally potent in vivo. It has further been shown that the removal of the amino terminal tryosine from GRF results in complete loss of bioactivity indicating that the active core of the molecule starts with the first amino terminal amino acid.
Growth in animals is believed to be regulated by a cascade of bio-regulatory molecules. Thus, the hypothalmus produces GRF which in turn acts upon the pituitary to cause release of growth hormone. The pituitary is maintained under negative feedback control by somatostatin and insulin growth factor (IGF). GRF has been found to be enormously active, exhibiting an ED.sub.50 of approximately 50 fmole/ml or 75 pg/ml and has been found to release micrograms/ml levels of growth hormone in the blood. Thus, GRF can be utilized therapeutically in most of the areas now considered candidates for treatment by growth hormone. Examples of such therapeutic uses include the treatment of pituitary dwarfism, diabetes resulting from abnormalities in growth hormone production, enhancement of wound healing, treatment of burns and retardation on the aging process. Due to its favorable potency compared to growth hormone itself, GRF will have major advantages in the agricultural field as well. Agricultural uses would include, for example, stimulating development of fowl or animals raised for meat so as to allow either marketing at an earlier time or else allow the farmer to produce a larger animal per equal time on feed to present methodology. In addition, GRF would be useful in stimulation of milk production in dairy cows and increasing egg production in chickens.
While GRF in its various forms is of a molecular size which would allow for synthesis by either conventional solid phase or solution phase peptide synthetic methods, it is believed that for economic, large scale production of these therapeutically valuable substances the use of recombinant DNA technology is preferred.
There are examples already known in the art for producing recombinant mammalian peptides utilizing genes synthesized by chemical methods. Thus, for example, in Science 198, page 1956 (Dec. 9, 1977) there is reported the production of recombinant human somatostatin in E. coli utilizing a chemically synthesized gene. This gene was fused to the E. coli beta-galactosidase gene on the plasmid pBR 322. Transformation of E. coli with the chimeric plasmid DNA lead to the synthesis of a polypeptide including the sequence of amino acids corresponding to somatostatin. Biologically active somatostatin was specifically cleaved from the chimeric protein by treatment with cyanogen bromide. This procedure is described in greater detail in U.K. Patent Application No. 2,007,675A. More recently substantially larger synthetic genes have been synthesized and cloned such as, for example, the gene for human leukocyte interferon.